I isolate neutrophils 4-5 times per week, so I guess you could call me an
expert. Neutrophil isolation is relativly easy, however they are extremely
sensitive cells. Unfortunatly I cannot provide you with a reference, however
I'll write out my protocol below. This protocol can be used with any mammalian
blood (I've done it with humans, mice and cats). But before I start I'll give
you some tips:
1) Do the whole thing at 4C, this will help limit activation
2) Avoid jaring/vibrating the tube, as this can activate the cells
3) Don't use glass - the cells will stick and not come off
This protocol purifies neutrophils using three steps:
1) Dextran sedimentation - this removes most of the RBC's. The RBC's sediment
to the bottom of the tube, while the leukocytes and lymphocytes remain
suspended in solution.
2) Hypotonic lysis - this removes any remaining RBC's and platlets.
3) Ficol sedimetntation - this separates mononuclear cells from neutophils.
The neutrophils sink to the bottom of the ficol, mononuclear cells remain at
the ficoll/isoalte interface.
I get 98% pure neutrophils or better using this protocol, with little to no
activation.
Heres the protocol:
1. Pipet 4 ml of ACD into a 50 ml conical tube and pour 20 ml of whole blood
down the side of the tube. Gently invert the tube several times to mix. (for
other volumes use 1ml ACD for every 5ml blood).
2. Pipet 12 ml (50% of blood volume) of 6% Dextran / 0.9% NaCl solution into
the ACD/blood mixture and invert 18-20 times to ensure adequate mixing. Pipette
the mixture into 4 15ml tubes (~10ml/tube). Let the four tubes stand at room
temperature for 45min - 1hr, or until separation is complete.
3. Return to the settling blood and pipet the yellowish supernatant into a 50
ml tube. Spin at 1150 rpm for 12 minutes at 4C using a low brake.
4. Discard the supernatant and resuspend in 12 ml of the ice-cold ddH2O. Suck
up and dispense repeatedly to break the pellet. After 20 seconds add add 4 ml
of 0.6 M KCl and mix several times. Dilute the solution to 50 ml with PBS.
Spin at 1300 rpm for 6 minutes at 4C using a high brake.
5. Repeat Step 4 1-2 times, until no RBC's remain.
6. Discard the supernatant, and resuspend the pellet in 2.5 ml of PBS.
7. Layer the cell suspension over 3 ml of Ficoll-Hypaque (Sigma 1077) in a 15
ml tube. Spin at 1500 rpm for 30 minutes at 4C using a low brake.
8. When the cells have finished spinning suck off the supernatant, using a
transfer pipet. Resuspend the pellet in 2 ml HBSS.
9. Determine the cell conentraiton using a hemocytometer, or other device.
Cells are noe ready for use. Neutrophils will remain viable suspended in HBSS
for about 4 hours at 4C, and about half that at room temperature. They can
also be resuspended in RPMI instead of HBSS, although this has been known to
activate them.
Here's how to make the solutions used in the protocol:
___________________________
ACD:
To 250ml ddH2O add:
7.36 gm Citric Acid
14.71 gm Sodium Citrate
9.91 gm Dextrose
Store at 4C
___________________
6% Dextran:
To 250ml ddH2O add:
15.00 gm Dextran (at least 100 000 MW)
2.25 gm NaCl
Store at 4?C
_______________________________
0.6M KCl:
To 250ml ddH2O add:
11.18 gm KCl
Store at 4C
_____________________________
Hope this helps
Bryan Heit
Immunology Research Group
University of Calgary
Calgary, Canada
